1Medizinische Fakultät Mannheim der Universität Heidelberg Abteilung für Herz- Kreislaufforschung Mannheim, Deutschland; 2Alexandra General Hospital Department of Clinical Therapeutics Athens, Griechenland; 3Universitätsklinikum Mannheim I. Medizinische Klinik Mannheim, Deutschland; 4Newcastle Biosciences Institute NE1 3BZ, Großbritannien
Introduction RNA-binding protein HuR may control the fate of several genes in a synchronized manner comprising an additional regulatory layer of gene expression. Here, we examined the role of HuR in human atherosclerosis.
Methods Single-cell and -nucleus RNA sequencing (scRNA, snRNA-seq) data were analyzed to compare cell-specific expression profiles of HuR and its target genes in murine or human atherosclerotic plaques. HuR individual-nucleotide cross-linking and immunoprecipitation equencing (iCLiP-seq) and RNA immunoprecipitation sequencing (RIP-seq) were applied to detect the HuR targetome in human transcriptome under basal and TNF-a pro-inflammatory conditions. Small interfering RNA (siRNA)-mediated HuR silencing, HuR overexpression, RNA stability assays and pharmacological HuR inhibition were used to determine the effect of HuR in the regulation of pro-atherosclerotic genes. Expression levels of HuR were measured in peripheral blood mononuclear cells derived from 543 individuals at risk for atherosclerotic heart disease and 175 patients with established chronic coronary syndromes (CCS). Structural and functional vascular measurements including intima-media thickness (IMT), carotid wall maximum thickness (maxWT) and number of atheromatous plaques by carotid and femoral artery ultrasonophaphy were used as surrogate markers of subclinical CVD. Study participants were followed for major adverse cardiovascular events (MACE).
Results Single cell analysis revealed increased HuR expression levels in vascular endothelium and infiltrated immune cells derived from a) Ldlr-/- mice treated with high-fat diet compared to chow diet, b) human carotid atherosclerotic plaques and c) diseased coronary arteries compared to controls. HuR iCLiP-seq and RIP-seq experiments revealed HuR binding sites in the 3’ untranslated region of several pro-atherosclerotic genes regulating local adhesion assembly, TGFBR-signaling, cell migration and differentiation. Silencing of HuR reduces while overexpression of HuR induces the expression of several pro-atherosclerotic genes in vascular endothelial cells (P<0.05 for all). Inhibition of transcription by actinomycin D showed that HuR controls the stability of pro-atherosclerotic genes including CCL2, CTSS, CXCL1, CXCL2 and SERPINE1 (P<0.05 for all). Pharmacological inhibition of HuR binding reduced the expression of targeted genes. In humans, HuR mRNA expression was increased in CCS compared to non-coronary artery disease (CAD) participants (p<0.001). Increased HuR expression was independently associated with the presence of CAD (OR=2.67 for highest vs. lower HuR tertiles), higher C-reactive protein (mean increase 23.6%) and mean carotid IMT after adjustment for traditional risk factors (P<0.05 for all). In the non-CAD population, increased HuR at baseline was prospectively associated with accelerated progression of subclinical atherosclerosis reflected as increased number of carotid and femoral plaques at follow-up (P<0.001) and a higher rate of increase in maxWT compared to patients at lower tertiles (P for interaction=0.045). Furthermore, high HuR concentrations were independently associated with higher MACE incidence across a median follow-up period of 48 months (higher versus lowest tertile: 7.4% vs. 1.27%, log rank test P=0.009).
Conclusion The stabilizing RNA-binding protein HuR controls the stability of several pro-atherosclerotic targets and predicts the progression of atherosclerosis and MACE in humans.